Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
PLoS One. 2021 Feb 26;16(2):e0247851. doi: 10.1371/journal.pone.0247851. eCollection 2021.
Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6-117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25-150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.
尿液无细胞 DNA（cfDNA）是一种有价值的非侵入性生物标志物，具有广泛的潜在临床应用，但在其最佳的预分析方法学方面，包括 DNA 提取步骤，尚未达成共识。由于其长度较短（大多数片段<100bp）且浓度较低（ng/mL），传统的基于硅胶的提取方法无法有效地回收尿液 cfDNA。为了最大限度地提高尿液 cfDNA 检测的灵敏度，我们开发了一种超灵敏的杂交方法，该方法使用固定在磁珠上的序列特异性寡核苷酸捕获探针来提高从大容量尿液样本中提取短 cfDNA 的效率。我们的杂交方法可以从 10ml 尿液中回收近 100%（95%置信区间：82.6-117.6%）的目标特异性 DNA，与片段长度无关（25-150bp），检测限≤5 拷贝双链 DNA（0.5 拷贝/mL）。将杂交与超短 qPCR 设计相结合，我们可以有效地捕获和扩增短至 25bp 的片段。我们的方法可以在单个 qPCR 孔中扩增 10ml 尿液中的 cfDNA，耐受样品组成的变化，并有效地去除非目标 DNA。我们的杂交方案改进了现有的基于硅胶的尿液 cfDNA 提取方法和以前的基于杂交的样品制备方案。两个关键创新点为我们的方法提供了强大的性能：双探针系统可回收双链 DNA 的两条链，以及双生物素化捕获探针，这通过在珠表面上实现最佳探针密度、提高探针-珠键的热稳定性以及消除内源性生物素的干扰，确保了一致的高回收率。我们最初为从尿液 cfDNA 中诊断结核病设计了杂交方法，但预计它将在尿液 cfDNA 靶标中具有通用性，并且可能对其他 cfDNA 样本类型和 cfDNA 以外的应用有用。为了使新用户能够使用我们的杂交方法，我们提供了详细的方案和设计新捕获探针的简单指南。
